1. Field of the Invention
The present invention relates to a method, test medium, and novel chromogenic compounds for quantitatively identifying and differentiating general coliforms and Escherichia coli. 
2. Description of the Related Art
Currently, in microbiology, the presence of indicator organisms is widely used to determine the quality of various products. For example, in the analysis of water, food and dairy products, the presence of members of the xe2x80x9ccoliformxe2x80x9d group as well as the presence of the bacterial species Escherichia coli are considered very significant quality indicators. Therefore, test methods to effective identify and enumerate these bacterial types are needed, and there is a continuing search for better, more accurate and simpler test methods in this area.
Numerous methods for determining, identifying and enumerating coliforms and E. coli currently exist, with varying degrees of accuracy and facility. Some test methods only indicate the presence or absence (P/A) of the organisms while some methods attempt to quantify the organisms in the test materials. Following are some of the current methods.
Violet Red Bile Agar (VRBA): This medium incorporates bile salts to inhibit non-coliforms. It also contains lactose with the pH indicator neutral red. As coliforms (especially E. coli) grow in the medium, the lactose is fermented with acid production and the neutral red in the area of the bacterial colony becomes a brick red color. Therefore, any colonies growing as a red color in 24-48 hours are considered to be coliforms. This medium is not easy to interpret and for E. coli quantification needs to be followed up by confirming tests such as brilliant green lactose broth fermentation or streaking on Eosin Methylene Blue Agar (EMBA). In spite of these shortcomings, VRBA is an approved method for testing dairy products.
The Most Probable Number (MPN) method: This method utilizes various broth (liquid) media in tubes. Samples to be tested are added in varying amounts to the broth media and after incubation, the tubes are checked for growth and gas formation. Estimates of the numbers (populations) of bacteria are determined from pre-existing tables. The method is in general use, but the results are given in a general range and therefore are riot very precise.
The Membrane Filter (MF) method: This method utilizes micropore filters through which samples are passed so that the bacteria are retained on the surface of the filter. It is used most often when bacterial populations are very small and a large sample is needed to get adequate numbers. The filter is then placed on the surface of a chosen medium, incubated and the bacterial colonies are counted and evaluated. This method is widely used and gives good results in general if combined with proper reagents and media, but is expensive and time-consuming. The MF method can be used well in combination with the new method described herein.
The Presence/Absence (P/A) test: This test, which involves the reagents O-nitrophenyl-xcex2-D-galactopyranoside (ONPG), a xcex2-galactosidase substrate and 4-methyl-umbelliferyl-xcex2-D-glucuronide (MUG), a xcex2-glucuronidase substrate, results in the determination of the presence or absence of general coliforms and E. coli. The test relies on the fact that generally all coliforms produce xcex2-galactosidase, but only E. coli strains produce xcex2-glucuronidase. If any coliforms are present, the broth medium turns a yellow color due to the activity of galactosidase enzyme on the ONPG material causing the release of a diffusible yellow pigment. If E. coli is present, the broth medium will demonstrate a blue fluorescence when irradiated with ultraviolet rays due to the breakdown of the MUG reagent with the release of the fluorogenic dye caused by the production of the glucuronidase enzyme. These reactions are very specific and allow both general coliforms and E. coli to be identified in a single test in a single sample. But, since both reagents produce diffusible pigments, the test has the disadvantage of not being directly quantitative for either bacterial type.
The reagent 5-bromo-4-chloro-3-indolyl-xcex2-D-galactopyranoside (X-gal) is a known test compound for identifying coliforms. When acted on by the xcex2-galactosidase enzyme produced by coliforms, X-gal forms an insoluble indigo blue precipitate. X-gal can be incorporated into a nutrient medium such as an agar plate, and if a sample containing coliforms is present, the coliforms will grow as indigo blue colonies. X-gal has the advantage over the compound ONPG, described above, in that it forms an insoluble precipitate, rather than a diffusible compound, thereby allowing the quantitative determination of coliforms.
Recently, a similar compound, 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide (X-gluc) has been developed for the identification of E. coli. When acted on by the xcex2-glucuronidase enzyme produced by E. coli, X-gluc forms an insoluble indigo blue precipitate. X-gluc has the advantages over the compound MUG, described above, in that it forms an insoluble precipitate, rather than a diffusible compound, thereby allowing the quantitative determination of E. coli. Further, it does not require the use of ultraviolet light. X-gluc and its use to identify E. coli are described in Watkins, et al, Appl. Environ. Microbiol. 54:1874-1875 (1988). A similar compound, indoxyl-xcex2-D-glucuronide, which also produces sharp blue colonies of E. coli, was described in Ley, et al, Can. J. Microbiol. 34:690-693 (1987).
X-gal and X-gluc have the disadvantage that they each contain the exact same chromogen and therefore they cannot be used together to identify and distinguish between both E. coli and general coliforms in a single test with a single sample. Both X-gal and X-gluc cause the formation of identically hued indigo blue colonies. A person using both reagents together would be able to quantitatively identify the total number of coliforms, the same as if X-gal were used alone, but would not be able to tell which of the colonies were E. coli and which were other coliforms besides E. coli. 
A method has now been found for quantitatively identifying and differentiating microorganisms having xcex2-galactosidase but not xcex2-glucuronidase activity and microorganisms having xcex2-glucuronidase activity, comprising the steps of combining a chromogenic xcex2-galactosidase substrate capable of forming an insoluble precipitate of a first color upon reacting with xcex2-galactosidase, a chromogenic xcex2-glucuronidase substrate capable of forming an insoluble precipitate of a second color contrasting with the first color upon reacting with xcex2-glucuronidase, and a nutrient base medium to form a test medium, inoculating the test medium with a sample to be tested for the presence of microorganisms, incubating the test medium, examining the test medium for the presence of colonies of the first color, such colonies being colonies of microorganisms having xcex2-galactosidase but not xcex2-glucuronidase activity, and the presence of colonies of the second color, such colonies being colonies of microorganisms having xcex2-glucuronidase activity, and enumerating the microorganisms having xcex2-galactosidase but not xcex2-glucurosidase activity and the microorganisms having xcex2-glucuronidase activity.
The advantages of the new method include the following. First, it allows the chromogenic differentiation between general coliforms, which have xcex2-galactosidase activity, and E. coli, which additionally have xcex2-glucuronidase activity, in the same test plate with the same sample. It is also quantitative so that exact counts of the numbers of viable organisms of each type are determined. This is much more meaningful than just a presence/absence test as levels of contamination can be determined. The new method does not require any special apparatus or equipment such as a UV light source or special filter apparatus. The new method is based on enzymatic reactions rather than fermentation reactions which are more difficult to interpret and less precise. Because the new method does not require inhibitors, there is the additional capability of quantifying the general microbial population along with general coliforms and E. coli. This is an important added feature.
A further aspect of this invention is the novel compound 6-chloroindolyl-xcex2-D-glucuronide, which forms an insoluble magenta precipitate when reacted upon by xcex2-glucuronidase that contrasts in color with the indigo blue precipitate formed by 5-bromo-4-chloro-3-indolyl-xcex2-D-galactopyranoside (X-gal) by the action of xcex2-galactosidase. 6-Chloroindolyl-xcex2-D-glucuronide may be used together with X-gal in the method of this invention. General coliforms grow in a medium containing 6-chloroindolyl-xcex2-D-glucuronide and X-gal as colonies having an indigo blue color whereas E. coli grow as colonies having a purplish or magenta color. Composition and test media having 6-chloroindolyl-xcex2-D-glucuronide and 5-bromo-4-chloro-3-indolyl-xcex2-D-galactoside for quantitatively identifying and differentiating general coliforms and E. coli are yet another aspect of this invention.
A further aspect of this invention is the novel compound 6-chloroindolyl-xcex2-D-galactoside, which forms an insoluble magenta precipitate when reacted upon by xcex2-galactosidase that contrasts with the indigo blue precipitate formed by 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide (X-gluc) or indoxyl-xcex2-D-glucuronide by the action of xcex2-glucuronidase. 6-Chloroindolyl-xcex2-D-galactoside may be used together with X-gluc or indoxyl-xcex2-D-glucuronide in the method of this invention. General coliforms grow in a medium containing 6-chloroindolyl-xcex2-D-galactoside and either X-gluc or indoxyl-xcex2-D-glucuronide as colonies having a magenta color whereas E. coli grow as colonies having an indigo blue color. Compositions and test media having 6-chloroindolyl-xcex2-D-galactoside and either 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide or indoxyl-xcex2-D-glucuronide for quantitatively identifying and differentiating general coliforms and E. coli are yet another aspect of this invention.